Therapeutic oligonucleotides targeting the human MDR1 and MRP genes

ABSTRACT

The present invention provides novel compositions and methods useful in cancer therapy for inhibiting the multidrug resistance phenotype, which often thwarts long-term chemotherapeutic regimens. The novel compositions of matter comprise oligonucleotides targeted to the human MDR1 and MRP genes, which inhibit expression of these genes, thereby rendering tumors and other forms of cancer more susceptible to the cytotoxic effects of chemotherapeutic agents. Oligonucleotides are also provided that inhibit the multidrug resistance phenotype by exerting an aptameric effect.

This is a continuation of U.S. patent application Ser. No. 08/487,141, filed Jun. 7, 1995, now U.S. Pat. No. 5,683,987 which is a continuation-in-part of U.S. patent application Ser. No. 08/379,180, filed Jul. 12, 1994, now abandoned, both of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to novel compositions and methods useful in cancer therapy for inhibiting the multidrug resistance phenotype, which is responsible for the failure of existing chemotherapeutic regimens to induce enduring remissions in the majority of cancer patients (Clarke et al., J. Natl. Cancer Inst. 84: 1506-1512, 1992). Specifically, the invention provides selected oligonucleotides (hereinafter "oligos"), and methods of use thereof, for inhibiting expression of genes responsible for the MDR phenotype, and for exerting aptameric inhibition of the MDR phenotype.

BACKGROUND OF THE INVENTION

The multidrug resistance phenotype is dependent upon the expression of molecular pumps that are capable of expelling chemotherapeutic agents from their site of action in cancer cells. These molecular pumps include, for example, the P-glycoprotein (hereinafter: P-gp) pump, and the multidrug resistance-associated protein (hereinafter: MRP) pump, which are encoded by the MDR1 and MRP genes, respectively.

The discussion set forth below illustrates the problems faced by clinical investigators seeking to improve cancer treatments. A number of references have been included to describe the general state of the art. Inclusion of these references is not an admission that such references represent prior art with respect to the present invention.

Multidrug resistance (hereinafter "MDR") is largely dependent on the expression of one or the other or both of two different genes. These genes encode transmembrane energy-dependent molecular "pumps" that expel a wide variety of anticancer agents from their site of action in tumors (Grant et al., Cancer Res. 54: 357-361, 1994; Riordan and Ling, Pharmacol. Ther. 28: 51-75, 1985). The normal functions of these molecular pumps is not well defined, but both are expressed by hematopoietic cells and P-gp has been shown to have a causal role in immunofunctioning (Gupta et al., J. Clin. Immunol. 12: 451-458, 1992). The MRP is a molecular pump initially found to be involved in multidrug resistance in lung cancer, and then later found to be expressed in other cancer types, while P-gp is a molecular pump long known to be involved in producing multidrug resistance in many tumor types (Chin et al., Adv. Cancer Res. 60: 157-180, 1993; Cole et al., Science 258: 1650-1654, 1992; Grant et al., Cancer Res. 54: 357-361, 1994; Krishnamachary and Center, Cancer Res. 53: 3658-3661, 1993; Zaman et al., Cancer Res. 53: 1747-1750, 1993).

Clinical studies in which P-gp inhibitors were administered prior to chemotherapy showed that such competitive inhibitors could increase the response of the tumors to the anticancer agents without causing an equivalent increase in toxicity to normal tissues (Marie et al., Leukemia 7: 821-824, 1993; Miller et al., J. Clin. Oncol. 9: 17-24, 1991; Pastan and Gottesman, Annu. Rev. Med. 42: 277-286, 1991; Raderer and Scheithauer, Cancer 72: 3553-3563, 1993; Sonneveld et al., Lancet 340: 255-259, 1992). Oligos designed to block the expression of MRP or P-gp have several features which should make them more clinically effective than any of the existing competitive inhibitors of P-gp or to any comparable inhibitors for MRP.

First, most chemical inhibitors used clinically to combat multidrug resistance have serious side effects unrelated to their ability to inhibit P-gp. In contrast, the phosphorothioate oligo, OL(1)p53, has been found to be essentially devoid of any toxicity when administered to patients (Bayever et al., Antisense Res. Dev. 2: 109-110, 1992; Antisense Res. Dev., in press, 1994). Similarly, this and other phosphorothioates have been shown to be nontoxic to a variety of animal species, even when given at high doses (Cornish et al., Pharmacol. Com. 3: 239-247, 1993; Crooke, Ann. Rev. Pharm. Toxicol. 32: 329-376, 1992; Iversen, Anti-Cancer Drug Design 6: 531-538, 1991). These findings show that at least some types of oligo have no acute toxicity per se when given systemically to animals or patients.

Second, some oligos, including phosphorothioates, have been shown often to have an RNAse-H dependent mechanism of action (Crooke, Ann. Rev. Pharm. Toxicol. 32: 329-376, 1992). RNAse-H enzyme activity is often expressed in clonogenic cells, while little or no activity is found in differentiated (non-proliferative) cells (Papaphilis et al., Anticancer Res. 10: 1201-1212, 1990; Crooke, Ann. Rev. Pharm. Toxicol. 32: 329-376, 1992). Because of this, blocking MRP or P-gp synthesis with oligos (as opposed to blocking their function by competitive inhibitors) should be relatively more effective in proliferating than in non-proliferating cells. Most normal cells that express P-gp or MRP are non-proliferating. For example, gastrointestinal crypt cells (stem cells) do not express P-gp, whereas the endstage (non-proliferating) luminal cells do (Chin et al., Adv. Cancer Res. 60: 157-180, 1993). Furthermore, once MRP or P-gp synthesis is blocked, the remaining membrane-associated drug-efflux pump of the parent cell would then be divided between the two daughter cells, reducing the effective amount of the molecular pump in the proliferating tumor cell population by one-half for each population doubling.

In addition, several recent papers report that ODNs not only are capable of blocking the expression of particular genes in vitro, but also are able to produce this effect in vivo. Some groups have successfully inhibited HIV gene expression (including tax) in human cells in xenogeneic transplant models (Kitajima et al., J. Biol. Chem. 267:25881-25888, 1992). Others have targeted genes in cancer cells, including c-myc, c-Ha-ras, NF-kB, c-myb, c-kit and bcr-abl. In each of these instances involving the administration of ODNs to treat animals with xenogeneic human cancers, the transplanted malignant cells were found to regress (Agrawal et al., Proc. Natl. Acad. Sci. 86:7790-7794, 1989; Proc. Natl. Acad. Sci. 88:7595-7599, 1991; Biro et al., Proc. Natl. Acad. Sci. 90:654-658, 1993; Gray et al., Cancer Res. 53:577-580, 1993; Higgins et al., Proc. Natl. Acad. Sci. 90:9901-9905, 1993; Ratajczak et al., Proc. Natl. Acad. Sci. 89:11823-11827, 1992; Wickstrom et al., Cancer Res. 52:6741-6745, 1992).

Furthermore, the Food and Drug Administration has approved several phosphorothioate antisense oligonucleotides for systemic administration to patients and for ex vivo treatment of hematopoietic stem cell grafts. These approvals include the now-completed OL(1)p53 phase I clinical trials (both systemic and ex vivo administered) which targeted transcripts of the p53 gene in patients with acute myeloid leukemia (Bayever et al., Antisense Res. Develop. 2: 109-110, 1992; Karp and Broder, Cancer Res. 54: 653-665, 1994). Thus, antisense oligonucleotides have the pharmacologic properties necessary for use as drugs.

There are six reports claiming reduced drug resistance in cultured cell lines following treatment with oligos targeting MDR-1 mRNA. Three of these (Vasanthakumar & Ahmed, Cancer Com. 1: 225-232, 1989; Rivoltini et al., Int. J. Cancer 46: 727-732, 1990; Efferth & Volm, Oncology 50:303-308, 1993) are totally unconvincing because they used oligos directed against mouse MDR-1 to treat human cells; in the corresponding human MDR-1 sequence, the longest matching nucleotide sequence was only 6 bases long. The paper by Thierry et al. (Biochem. Biophys. Res. Comm. 190: 952-960, 1993) reports no oligo with a sequence which matches the human MDR-1 gene, but this problem is apparently due to typing errors (personal communication from A. Thierry). Thierry's 15-mer that gave 95% inhibition of MDR-1 expression did so only when encapsulated in liposomes; this was associated with a 4-fold increase in sensitivity of the tumor cells to doxorubicin (Thierry et al., Biochem. Biophys. Res. Comm. 190: 952-960, 1993); when administered without liposomes, inhibition of MDR1 expression was 40% of control values. Furthermore, the calculated melting temperature for Thierry's 15-mer is less than 28° C., suggesting that at body temperature the amount of oligo bound is very low.

The most compelling papers in this group are by Jaroszewski et al. (Cancer Comm. 2: 287-294, 1990) and Corrias and Tonini (Anticancer Res. 12: 1431-1438, 1992). Both teams found inhibition with only one out of five oligos. Jaroszewski et al. (Cancer Comm. 2: 287-294, 1990) describe one phosphorothioate (which is being designated "Cohen(1)mdr" herein) that gave 25% reduction in P-gp expression at 15 μM and 33% reduction at 30 μM when incubated with MCF-7/ADR breast cancer cells for 5 days. This reduction in P-gp expression was associated with a small increase in the doxorubicin sensitivity of the cells (20% increase in cell death when 10 μM of the oligo was used. Corrias & Tonini (Anticancer Res. 12: 1431-1438, 1992) report a phosphodiester oligo that gave only a slight reduction in P-gp (data not shown herein) at 30 μM when incubated with doxorubicin-resistant colon adenocarcinoma cells for 36 hours. The reduction in P-gp expression was associated with a significant increase in the in vitro sensitivity of the cells to the cytotoxic effects of doxorubicin (80% and 53% dose reductions in IC₅₀, respectively; the IC₅₀ being the inhibitory concentration of a chemotherapeutic agent (e.g., doxorubicin) which causes a 50% inhibition in cellular proliferation).

It is, therefore, a principal object of the present invention to provide MDR-oligos or MRP-oligos that target the genes encoding P-gp or MRP, respectively, or their RNA transcripts, in order to specifically and effectively sensitize clonogenic multidrug-resistant tumor cells to chemotherapeutic agents. It is another object of the present invention to provide oligos which will sensitize tumor cells much more efficiently than they do normal cells which express these same molecular pumps. As the foregoing discussion highlights, oligonucleotides effective for these purposes heretofore have been unavailable.

There is growing evidence that certain protein kinases, such as protein kinase A (PKA), and protein kinase C (PKC) in particular, are involved in the activation of the forms of drug resistance which depend on the expression of molecules producing multidrug resistance, such as, for example, P-glycoprotein, MRP, pi-class glutathione S-transferase, gamma-glutamylcysteine (Gekeler et al., Biochem. Biophys. Res. Comm. 205: 119, 1995; Grunicke et al., Ann. Hematol. 69 (Suppl 1): S1-6, 1994; Gupta et al., Cancer Lett. 76: 139, 1994), or the transmembrane pump capable of expelling glutathione conjugates from their site of action in cells (such as the GS-X pump (Ishikawa et al., J. Biol. Chem. 269: 29085, 1994).

These second-messenger pathways typically appear to be more active in drug resistant cancer cells compared to their drug sensitive counterparts. These pathways promote the expression of various drug resistance phenotypes by causing the up-regulation of a very small number of specific transcriptional regulators, including AP-1, that presumably control the activation of molecules involved in producing drug resistance in cancer cells (Grunicke et al., Ann. Hematol. 69(Suppl 1): S1-6, 1994). For example, activation of the ras oncogene in malignant cells is one of the ways that PKC and, in turn, multidrug resistance, can be up-regulated in tumor cells. Hence, the realization that what has been considered to be multiple discrete mechanisms for drug resistance share common activation pathways opens a new set of possibilities for broad spectrum therapeutic interventions.

Thus, if inhibitors of these second messenger pathways could be found, they should be of use for treating a wider variety of multidrug resistance phenotypes in cancer than agents designed to inhibit the function or expression of a single molecular species involved in drug resistance (Grunicke et al., Ann. Hematol. 69(Suppl 1): S1-6, 1994; Christen et al., Cancer Metastasis Rev. 13: 175, 1994). In addition, inhibitors of particular PKC isoenzymes, or of PKA, or molecular regulators up- or down-stream of these enzymes, should find a variety of other applications where these protein kinases are known to play an important role, including the treatment of viral infections, AIDS, Alzheimer's Disease, and conditions where immunosuppression is important such as in autoimmune diseases, transplantation-related reactions and inflammatory reactions.

Stein et al. (Biochemistry 32: 4855, 1993) discovered that both a 15-mer phosphodiester homopolymer of thymidine and a 28-mer phosphorothioate homopolymer of cytidine could inhibit the B1 isoenzyme of PKC, with the result that pinocytosis and cellular uptake of macromolecules was inhibited. For the 28-mer phosphorothioate, the IC₅₀ for directly inhibiting purified PKC-β1 activity was 1 μM; complete suppression required nearly 40 μM.

Conrad et al (J. Biol. Chem. 269: 32051, 1994) have shown that certain RNA aptamers can inhibit the βII isoenzyme of PKC. These RNA aptamers were selected from a pool of RNA molecules that contained a 120-nucleotide randomized region. PKC-βI is an alternatively-spliced variant of PKC-βII.

Schuttze et al (J. Mol. Biol. 235: 1532, 1994) have analyzed in detail the three-dimensional solution structure of the thrombin-binding DNA aptamer d(GGTTGGTGTGGTTGG). This aptamer binds to thrombin and inhibits its activity in the chain of reactions that lead to blood clotting. The authors conclude that "knowledge of the three-dimensional structure of this thrombin aptamer may be relevant for the design of improved thrombin-inhibiting anti-coagulants with similar structural motifs."

Using the human KM12L4a colon cancer cell line, Gravitt et al. (Biochem. Pharmacol. 48: 375, 1994) discovered that the agent thymeleatoxin (which stimulates the phorbol ester-responsive PKC isoenzymes -α, -βI, -βII and -gamma) induces multidrug resistance. Since this cell line expresses only the PKC-α isoenzyme, it is clear that PKC-α lies in a second messenger pathway that can up-regulate multidrug resistance.

Fan et al (Anticancer Res. 12: 661, 1992) presented data showing that the expression of rat brain PKC-βI confers a multidrug resistance phenotype on rat fibroblasts.

Thus, it is another object of the present invention to provide oligonucleotides that inhibit various MDR phenotypes in cancer cells by exerting an aptameric effect.

SUMMARY OF THE INVENTION

In accordance with the present invention, there are provided novel oligonucleotides targeting the human MDR1 gene or the human MRP gene or their transcripts which are uniquely effective in inhibiting multidrug resistance in human cancer cells. Administration of these oligos to patients having multidrug resistant cancer is done for the purpose of increasing sensitivity of the cancer(s) to the cytotoxic effects of therapeutic agents that would normally be expelled from their site of action in the tumor cells by the MRP or P-gp molecular pumps. In addition, the oligos may be administered to a patient with cancer or a premalignant syndrome to prevent the development of multidrug resistance in the patient's tumor. The oligos may be used alone or in combination with certain chemical inhibitors which exhibit an inhibitory effect on the MRP or P-gp pumps. The oligos may also be used in combination with chemotherapeutic drugs to purge bone marrow or peripheral stem cell grafts of malignant cells or non-malignant mononuclear cytotoxic effector cells. The oligos may be administered to patients receiving an organ transplant, or patients with autoimmune diseases, as an immunosuppressive agent alone or with other MRP or P-gp inhibitors or with cytotoxic or cytostatic drugs. The oligos to MDR1 herein described are much more effective than other oligos presently known. Oligos directed toward inhibiting expression of the MRP gene have not been described previously, insofar as is known.

Furthermore, the present inventor has found that MRP-oligos have activity for reversing multidrug resistance phenotype in non-lung cancer.

"Prototype oligos" have been designed, synthesized and used to confirm in in vitro experiments that indeed the nucleotide target sequences for oligo binding indicated in Tables 1 through 3 ("hotspots") within the MRP and MDR1 gene sequences or transcript sequences are particularly suitable for the practice of the present invention. Variant oligos with suitable physical properties have also been designed which target the same general areas of the MDR1 or MRP sequences as the prototype oligos. Such variant oligos, described herein in Tables 4 and 5 are expected to also have utility for the same therapeutic purposes as the prototype oligos.

Also disclosed are a set of oligonucleotide sequences which can dramatically reverse the multidrug resistance phenotype exhibited by cancer cells, even though the oligo sequences are such that they are not complementary to any known human gene. They must act, therefore, by interfering with the function of some key molecule needed for the production of the multidrug resistance phenotype. This type of phenomenon is generally known as an "aptameric effect." The oligonucleotides exhibiting this specific aptameric effect (hereinafter "MDR-aptamer" oligos) are highly active in vitro at concentrations below 1 μM. The degree to which these MDR-aptamers reverse the multidrug resistance phenotype is positively correlated with the degree to which, by themselves, they inhibit the in vitro proliferation of drug-resistant cancer cells. These MDR-aptamers do not have a major drug sensitizing effect on drug sensitive cells and they do not significantly inhibit the proliferation of such cells. Similarly, some MDR- and MRP-Oligos exhibit both an antisense effect on MDR1 or MRP expression and (to verying degrees) an MDR-aptameric effect. These MDR-aptamers can serve a variety of purposes, including being used: (1) to treat cancer patients, particularly those with multidrug resistant cancer, in order to sensitize the tumor to chemotherapeutic agents; (2) as probes to discover the critical molecular target in cells (to which they bind) required for the maintenance of the multidrug resistance phenotype; and (3) as prototype MDR-aptamers in structural studies for the further development of oligos of this type for clinical use as therapeutic agents.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1. Graph of predicted ³ H-TdR uptake count (Y-axis) as a function of Log₁₀ (dose of drug) (X-axis) for selected mdr oligonucleotides. The graph plots the estimated functions (curved lines) associating Log₁₀ (Vincristine dose) with expected "³ H-TdR counts" for each oligo shown in the figure.

DETAILED DESCRIPTION OF THE INVENTION

In preferred embodiments of the invention, the oligos are administered systemically to cancer patients, either in combination with chemotherapeutic agents, in order to potentiate the elimination of multidrug resistant tumor cells from the body of the host; or alone, to prevent development of multidrug resistant phenotype, or with chemotherapeutic agents to purge malignant cells from bone marrow or peripheral stem cell grafts. The oligos also may be administered as an immunosuppressive agent.

The list of chemotherapeutic agents to be used in association with the prototype or variant oligos of the present invention is selected from but not limited to a list that comprises: (1) the vinca alkaloids: including vincristine, vinblastine, vindesine; (2) the anthracyclines, including daunorubicin, doxorubicin, idarubicin; (3) the epipodophyllotoxins, including VP-16 (etoposide) and VM-26 (teniposide); and (4) miscellaneous: steroids, mitomycin C, taxol, actinomycin D, melphalan.

The prototype or variant oligos may be used alone to inhibit P-gp or MRP function, or in combination with non-oligo inhibitors of these molecular pumps, selected from but not limited to a list comprising: PSC-833 (cyclosporin D analog), verapamil, cyclosporin A, FK506, tamoxifen, megestol, and novobiocin and its analogs.

The prototype or variant oligos may be used alone or in combination with the aforementioned agents to treat any human cancer which expresses a multidrug resistant phenotype, or to prevent the development of a multidrug resistance phenotype, including those types of human cancer selected from but not limited to a list comprising: breast cancer, lung cancer, colon cancer, liver cancer, renal cancer, pancreatic cancer, prostate cancer, ovarian cancer, cervical cancer, uterine cancer, bladder cancer, brain cancer, adrenal cancer, multiple myeloma, ear-nose-throat cancers (including esophageal, laryngeal, pharynx), leukemia, lymphoma, sarcoma and carcinoid tumors. This listing is included for the purpose of illustration only, and is not meant to limit the practice of the present invention.

The present invention encompasses oligonucleotides and oligonucleotide analogs which are complementary to selected target sites of MDR1 or MRP, and transcripts thereof. These target sites are sometimes referred to herein as "hotspots." Using a computer program, such as "Oligo" (Richik & Rhoades, Nucl. Acids Res. 17: 8543, 1989) and a reference such a Genbank, these hotspots were selected on the basis of their unique sequence (i.e., having high sequence homology with members of the gene family, but less than 85% homology with genes and RNA transcripts outside the gene family) and various physical parameters desirable for the antisense oligonucleotides to be produced. In preferred embodiments of the invention, oligos which are targeted to these "hotspots" possess the following characteristics: (1) length between about 10 and 40 bases, with a preferred range of about 15-30 and a particularly preferred range of 17-26; (2) negligible self-interaction (self-dimers and hairpins) under physiological conditions; (3) melting temperature ≧40° C. under physiological conditions; and (4) no more than 40% of the oligo composed of run of guanines or cytosines.

Preferred hotspots, prototype oligonucleotides and size variants thereof are set forth herein. These hotspots and oligos are disclosed with reference to specific MDR1 or MRP nucleotide sequences from the Genbank library. It will be appreciated by persons skilled in the art that variants (e.g., allelic variants) of these sequences exist in the human population, and must be taken into account when designing and/or utilizing oligos of the invention. Accordingly, it is within the scope of the present invention to encompass such variants, either with respect to the preferred hotspot disclosed herein or the oligos targeted to specific locations on the respective genes or RNA transcripts. With respect to the inclusion of such variants, the term "substantially the same as" is used herein to refer to various specific nucleotide sequences and variants thereof that would occur in a human population. Additionally, the term "substantially the same as" refers to oligo sequences that may not be perfectly matched to a target sequence, but the mismatches do not materially affect the ability of the oligo to hybridize with its target sequence under the conditions described.

The subject of the present invention is the nucleotide sequence of the disclosed oligos (listed in Tables 1 through 5) in association with a chemical backbone, the backbone selected from, but not limited to, a list consisting of the following types (reviewed in Neckers et al., Crit. Rev. Oncogen. 3: 175-231, 1992): phosphorothioates, dithioates, methylphosphonates, phosphodiesters, morpholino backbones, polyamide backbones, and any combination of the aforementioned backbone types, including, for example, phosphorothioate-capped phosphodiesters. The backbones may be unmodified, or they may be modified to incorporate a ribozyme structure, or a pendant group. Additionally, 2'-O-methyl (ribose-modified) oligos are suitable for the practice of the invention. The 2'-o-methyl sugar modification can be associated with any of the backbone linkages, including phosphorothioates, and the modification can be limited to the ends of the oligonucleotide. The oligos may also be associated with a carrier or vehicle such as liposomes or micelles, although other carriers could be used, as would be appreciated by one skilled in the art. Such carriers are used to facilitate the cellular uptake and/or targeting of the oligo, and/or improve the oligo's pharmacokinetic and/or toxicologic properties.

                                      TABLE 1     __________________________________________________________________________     Preferred 20-mer, 22-mer and 26-mer MDR-oligo nucleotide sequences     within targeting hotspots     SEQ ID  Nucleotide                  OLIGO (Trivial     ID  HOT-             Staiting                  Name) and variants                            OLIGO SEQUENCE     NO. SPOT             Position*                  of same length                            (5'--->3')     __________________________________________________________________________     1   1   488  OL(6)mdr prototype                            CCCACGCCCC GGCGCTGTTC     2       496  OL(6A)mdr GTGCTCAGCC CACGCCCCGG     3   2   517  OL(16)mdr prototype                            GGCAAAGAGA GCGAAGCGGC     4       518  OL(16A)mdr                            TGGCAAAGAG AGCGAAGCGG     5       540  SJ(34)mdr prototype                            TCGAATGAGC TCAGGCTTCC     6       542  SJ(34A)mdr                            TCGAATGAGC TCAGGCTT     7       540  SJ(34B)mdr                            ACTCGAATGA GCTCAGGCTT CC     8       533  SJ(34C)mdr                            AGCTCAGGCT TCCTGTGGCA     9       543  SJ(34D)mdr                            CGAATGAGCT CAGGCT     10  3   664  5(1)mdr prototype                            CCCTACCTCG CGCTCCTTGG AACGGC     11      688  OL(10)mdr GCTCCCAGCT TTGCGTGCCC     12  4   884  OL(12)mdr prototype                            GCGCGCTCCG GGCAACATGG     13      881  OL(12A)mdr                            CGCGCTCCGG GCAACATGGT CC     14      885  OL(12B)mdr                            CGCGCTCCGG GCAACATG     15      881  OL(12C)mdr                            CTCCGGGCAA CATGGTCC     16      941  OL(15)mdr prototype                            TGCTTCCTCC CACCCACCGC     17      937  OL(15A)mdr                            TCCTCCCACC CACCGCCCGC     18      938  OL(15B)mdr                            TTCCTCCCAC CCACCGCCCG     19      939  OL(15C)mdr                            CTTCCTCCCA CCCACCGCCC     20      940  OL(15D)mdr                            GCTTCCTCCC ACCCACCGCC     21  5   1000 OL(5)mdr prototype                            TCTGGACTTT GCCCGCCGCC     22      1001 OL(5A)mdr TTCTGGACTT TGCCCGCCGC     23      1002 OL(5B)mdr GTTCTGGACT TTGCCCGCCG     24      1003 OL(5C)mdr CGTTCTGGAC TTTGCCCGCC     25  6   1125 OL(1)mdr prototype                            GCTCCTCCAT TGCGGTCCCC     26      1123 OL(1B)indr                            GCTCCTCCAT TGCGGTCCCC TT     27      1125 OL(1C)mdr TCTTTGGTCC TCCATTGCGG TCCCC     28      1125 OL(1Q)mdr TTTGCTCCTC CATTGCGGTC CCC     29      1125 OL(1W)mdr TCCTCCATTG CGGTCCCC     30      1123 OL(1Wa)mdr                            CTCCATTGCG GTCCCCTT     31      1125 OL(1Wb)mdr                            CTCCATTGCG GTCCCC     32      1121 OL(1Wc)mdr                            CCATTGCGGT CCCCTTCA     33      1127 OL(1X)mdr GCTCCTCCAT TGCGGTCC     34      1122 OL(1A)mdr CCTCCATTGC GGTCCCCTTC     35  7   1688 OL(2)mdr prototype                            GCAACCAGCA CCCCAGCACC     36      1691 OL(2A)mdr GCAGCAACCA GCACCCCAGC     37  8   5996 OL(3)mdr prototype                            TGCCCACCAG AGCCAGCGTC     38      6278 OL(15)mdr prototype                            GCCTCCTTTG CTGCCCTCAC GA     39  9   6551 SJ(36)mdr prototype                            CCAGGGCTTC TTGGACAACC TA     __________________________________________________________________________      *The nucleotide starting position for targeting MDR1gene transcripts is      based on the GenBank entries HUMMDR1A01 through HUMMDR1A26, considered as      a single linear sequence, with Number 1 position being the most 5prime      nucleotide of the HUMMDR1A01 GenBank entry. The Nucleotide Starting      Positions in this Table represent the most 5prime nucleotide of the      corresponding sense sequence.

Particularly preferred for practice of the invention are oligos that hybridize to the hotspots listed below (or substantially equivalent variants) for MDR-1, Genbank reference No. HUMMDR1-AO1 through -AO26, taken consecutively (Chin et al., Mol. Cell. Biol. 9: 3808, 1989; Chen et al., J. Cell. Biochem. 265: 506, 1990).

Hot-spot 6: Range of bases includes positions 1121-1158

(bases numbered as per footnote to Table 1);

sequence below (Sequence I.D. No. 102) is coding strand:

5'-TGAAGGGGACCGCAATGGAGGAGCAAAGAAGAAGAACT-3'

Hot-spot 2: Range of bases includes positions 540-564;

sequence below (Sequence I.D. No. 103) is coding strand:

5'-GGAAGCCTGAGCTCATTCGAGTAGC-3'

Hot-spot 3: Range of bases includes positions 685-708;

sequence below (Sequence I.D. No. 104) is coding strand:

5'-AGGGGCACGCAAAGCTGGGAGCT-3'

Hot-spot 4: Range of bases includes positions 881-904:

sequence below (Sequence I.D. No. 105) is coding strand:

5'-GGACCATGTTGCCCGGAGCGCGCA-3'

Table 1A describes several sequence variants of antisense oligos in Hotspot 3 of the MDR1 gene. Table 1B describes very similar oligos that bind near the translational start site of the mRNA. Oligo "5(1C)mdr" (SEQ ID NO:94) binds to an upstream splice junction site, while oligo "5(1G)mdr" (SEQ ID NO:98) binds to a site just upstream of the start codon. Interestingly, nearly identical sequences (only 1 base difference in 22) exist in both the genomic and the spliced cDNA versions of the gene (starting positions #666 and #405, respectively). The fourth position guanine in SEQ ID NO:98 makes a perfect match with the "AUG" start site region, while a fourth-position thymidine in SEQ ID NO:94 makes a perfect match precisely with the upstream exon 1B/intron 1 splice junction. A variant of these oligos, synthesized with a fourth position inosine base (such as, for example, oligo "5(1J)mdr", SEQ ID NO:101) should be able, therefore, to bind equally to transcripts from either site.

                                      TABLE 1A     __________________________________________________________________________     Additional sequence variants in the Hotspot 3 region of the mdr1 gene     SEQ     Nucleotide                  Oligo     ID  HOT-             Starting                  Trivial                       Oligo Sequence     NO. SPOT             Position.sup.1                  Name (5'-->3')     __________________________________________________________________________     92  3   673  5(1A)mdr                       CGTGCCCCTA CCTCGCGCTC CT     93  3   666  5(1B)mdr                       CCCTACCTCG CGCTCCTTGG AACG     94  3   666  5(1C)mdr                       CCCT.sup.2 ACCTCG CGCTCCTTGG AA     95  3   671  5(1D)mdr                       CGTGCCCCTA CCTCGCGCTC CTTG     96  3   677  5(1E)mdr                       CGTGCCCCTA CCTCGCGC     __________________________________________________________________________

                                      TABLE 1B     __________________________________________________________________________     Additional sequence variants in the upstream splice junction site     SEQ     Nucleotide                  Oligo     ID  HOT-             Starting                  Trivial                       Oligo Sequence     NO. SPOT             Position.sup.4                  Name (5'-->3')     __________________________________________________________________________     97  --  408  5(1F)mdr                       TCCCGACCTC GCGCTCCT     98  --  403  5(1G)mdr                       CCCG.sup.2 ACCTCG CGCTCCTTGG AA     99  --  405  5(1H)mdr                       CCATCCCGAC CTCGCGCTCC TTGG     100 --  403  5(1I)mdr                       CCATCCCGAC CTCGCGCTCC TTGGAA     101 --  405  5(1J)mdr                       CCCI.sup.3 ACCTCG CGCTCCTTGG     __________________________________________________________________________      .sup.1 The nucleotide starting position for targeting MDR1gene transcript      is based on the GenBank entries HUMMDR1A01 through HUMMDR1A26, considered      as a single linear sequence, with Number 1 position being the most 5prime      nucleotide of the HUMMDR1A01 GenBank entry. The Nucleotide Starting      Positions in this Table represent the most 5prime nucleotide of the      corresponding sense sequence.      .sup.2 If an inosine base is placed in this fourth base position, then th      resulting ODN would effectively be a perfect match with both binding      sites.      .sup.3 This variant sequence contains an inosine base substituted at the      fourth position where the singlebase variation between SEQ ID NO: 94 and      SEQ ID NO: 98 exits.      .sup.4 The nucleotide starting position for targeting mdr1 mRNA is based      on GenBank entry HUMMDR1/M14758.

                                      TABLE 2     __________________________________________________________________________     Preferred 23-mer MDR-oligo nucleotide sequences within targeting     hotspots.     SEQ     Nucleotide                  OLIGO (Trivial     ID  HOT-             Starting                  Name) and variants                           OLIGO SEQUENCE     NO. SPOT             Position*                  of same length                           (5'--->3')     __________________________________________________________________________     40  10  670  PA(1)mdr prototype                           GCGGGAGGTG AGTCACTGTC TCC     41      670  AP(1)mdr GGAGACAGTG ACTCACCTCC CGC     __________________________________________________________________________      *The nucleotide starting position for targeting the MDR1gene is based on      the GenBank Entry 105674 "Hummdr1B", and represents the most 5prime      nucleotide of the corresponding sense sequence.

                                      TABLE 3     __________________________________________________________________________     Preferred 20-mer and 26-mer MRP-oligo nucleotide sequences within     targeting hotspots     SEQ     Nucleotide                  OLIGO (Trivial     ID  HOT-             Starting                  Name) and variants                            OLIGO SEQUENCE     NO. SPOT             Position*                  of same length                            (5'--->3')     __________________________________________________________________________     42  1   24   5(3)MRP prototype                            CGGCGGCGGC GGCGCAGGGA GCCGGG     43  2   169  5(2)MRP prototype                            CGGTGGCGCG GGCGGCGGCG GGCACC     44  3   220  OL(14)MRP prototype                            GCGGGTCGGA GCCATCGGCG     45      222  OL(14A)MRP                            GAGCGGGTCG GAGCCATCGG     46      223  OL(14B)MRP                            AGAGCGGGTC GGAGCCATCG     47      225  OL(14C)MRP                            CCAGAGCGGG TCGGAGCCAT     48  4   1210 OL(5)MRP prototype                            CTGCGGCCCG GAAAACATCA     49  5   2114 OL(2)MRP prototype                            CGGTGATGCT GTTCGTGCCC     50      2101 OL(2A)MRP CGTGCCCCCG CCGTCTTTGA     51      2102 OL(2B)MRP TCGTGCCCCC GCCGTCTTTG     52      2103 OL(2C)MRP TTCGTGCCCC CGCCGTCTTT     53      2104 OL(2D)MRP GTTCGTGCCC CCGCCGTCTT     54      2105 OL(2E)MRP TGTTCGTGCC CCCGCCGTCT     55      2106 OL(2F)MRP CTGTTCGTGC CCCCGCCGTC     56      2107 OL(2G)MRP GCTGTTCGTG CCCCCGCCGT     57      2108 OL(2H)MRP TGCTGTTCGT GCCCCCGCCG     58      2109 OL(2I)MRP ATGCTGTTCG TGCCCCCGCC     59      2110 OL(2J)MRP GATGCTGTTC GTGCCCCCGC     60  6   2516 OL(6)MRP prototype                            GGGCCAGGCT CACGCGCTGC     61      2519 OL(6A)MRP GCCCGGGCCA GGCTCACGCG     62  7   2848 OL(3)MRP prototype                            CCCTGGACCG CTGACGCCCG     63      2834 OL(3A)MRP CGCCCGTGAC CCCGTTCTCC     64  8   3539 OL(8)MRP prototype                            GCGGGATGAT GATGGCGGCG     65      3538 OL(8A)MRP CGGGATGATG ATGGCGGCGA     66      3540 OL(8B)MRP GGCGGGATGA TGATGGCGGC     67      3541 OL(8C)MRP GGGCGGGATG ATGATGGCGG     68      3542 OL(8D)MRP GGGGCGGGAT GATGATGGCG     69      3543 OL(8E)MRP AGGGGCGGGA TGATGATGGC     70      3528 OL(8F)MRP ATGGCGGCGA TGGGCGTGGC     71      3529 OL(8G)MRP GATGGCGGCG ATGGGCGTGG     72      3530 OL(8H)MRP TGATGGCGGC GATGGGCGTG     73      3531 OL(8I)MRP ATGATGGCGG CGATGGGCGT     74      3532 OL(8J)MRP GATGATGGCG GCGATGGGCG     75      3533 OL(8K)MRP TGATGATGGC GGCGATGGGC     76  9   4154 OL(4)MRP prototype                            CGATGCCGAC CTTTTCTCC     77      4160 OL(4A)MRP GCCCCACGAT GCCGACCTTT     78      4161 OL(4B)MRP CGCCCCACGA TGCCGACCTT     79      4162 OL(4C)MRP CCGCCCCACG ATGCCGACCT     80      4163 OL(4D)MRP TCCGCCCCAC GATGCCGACC     81  10  4933 3(3)MRP prototype                            TGGCGGTGGC TGCTGCTTTG     82      4936 3(3A)MRP  GGATGGCGGT GGCTGCTGCT     83      4937 3(3B)MRP  CGGATGGCGG TGGCTGCTGC     84  11  4637 OL(15)MRP prototype                            CCGGTGGGCG ATGGTGAGGA CG     __________________________________________________________________________      *The nucleotide starting position for targeting MRPgene trancripts is      based on the GenBank Entry #L05628: HUMMRPX", and represents the most      5prime nucleotide of the corresponding sense sequence.

Particularly preferred for practice of the invention are oligos that hybridize to the hotspots listed below (or substantially equivalent variants thereof) for MRP, Genbank reference No. HUMMRPX/L05628 (Cole et al., Science 258: 1650, 1992).

Hot-spot 1: Range of bases includes postions 3528-3566

(numbered as per footnote to Table 3);

Sequence below (Sequence I.D. No. 106) is coding strand:

5'-GCCACGCCCATCGCCGCCATCATCATCCCGCCCCTTGGC-3'

Hot-spot 2: Range of bases includes positions 2469-2538;

sequence below (Sequence I.D. No. 107) is coding strand:

5'-CGGACAGAGATTGGCGAGAAGGGCGTGAACCTGTCT-GGGGGCCAGAAGCAGCGCGTGAGCCTGGCCCGGG-3'

Hot-spot 3: Range of bases includes positions 1198-1242;

sequence below (Sequence I.D. No. 108) is coding strand:

5'-TCCACGACCTGATGATGTTTTCCGGGCCGCAGATCTTAAAGTTGC-3'

Hot-spot 4: Range of bases includes positions 2805-2896;

sequence below (Sequence I.D. No. 109) is coding strand:

5'-GCCAGCACAGAGCAGGAGCAGGATGCAGAGGAGAACGGGGTCACGGGCGTCA-GCGGTCCAGGGAAGGAAGCAAAGCAAATGGAGAATGGGAT-3'

                  TABLE 4     ______________________________________     Preferred Size Variants of MDR-Oligos at different starting positions     in the MDR1 gene sequence hotspots     HOT-    Nucleotide      Oligo Size Variants     SPOT    starting position                             (nucleotide length)*     ______________________________________     1       460             25.24.23.22             461             25.24.23.22             462             25.24.23.22             463             25.24.23.22.21             464             25.24.23.22.21             465             25.22.21.19             466             25.21.19             467             25.19.18             468             25.19.18.17             469             25.18.17             470             17             486             17             488             20             496             20.17             497             17             498             17     2       517             25.24.23.22.21.20.19.18.17             518             25.24.23.22.21.20.19.18             519             24.23.22.21.19.18             520             24.23.22.21             521             22.21             522             21             523             21     2       540             25,24,23,22,21,20,19,18             541             23,22,21,20,19,18             542             22,21,20,19,18     3       663             25.24.23.22.21.19             664             26.25.24.23.22.21.19.18             665             25.24.23.22.21.17             666             25.24.23             667             25.24.23             668             25.24.23.22             669             25.24.23.22             670             25.24.23.22.21             671             23.21             672             23.21             673             21             674             21             676             19             677             18             678             17             680             21.19.18.17             681             19.18.17             682             19.18.17             683             21.18.17             684             21.17             685             21             686             21.17             687             21.19.17             688             17     4       879             25,24,23,22             880             24,23,22             881             23,22             882             22             883             21             884             20             885             19             912             21.19.18             913             19.18             914             19.18             915             18             937             25.24.23.22.21.20.19.18.17             938             25.24.23.22.21.20.19.18.17             939             25.24.23.22.21.20.19.18.17             940             25.24.23.22.21.20.19.18.17             941             25.24.23.22.21.20.19.18.17             942             24.23.22.21.19.18.17             943             24.23.22.21.19.18.17             944             22.21             945             21.19.18             946             19.18             947             19.17     5       981             22.21.19.18.17             982             21.19             983             19             984             19             985             18             986             17             995             23.22.19.18.17             996             22.18.17             997             17             998             19.18.17             999             24.23.22.18.17             1000            24.23.22.21.20.19.18.17             1001            22.21.20.19.18.17             1002            21.20.19.18.17             1003            20.19.18.17             1004            19.18.17             1005            24.23.19.18             1006            23.18             1008            25.24.23             1009            25.24.23             1010            25.24.23             1011            25.24.23             1012            25.24.23             1013            25.24.23             1014            25.24.23             1015            25.23             1031            22.21             1032            21.19             1033            19.18.17             1034            19.17             1055            17             1088            25             1089            25             1090            25             1091            25             1092            25             1093            25             1094            25     6       1121            25.24.23.22.21.19.18.17             1122            25.24.23.22.21.20.19.18.17             1123            25.24.23.22.21.20.19.18.17             1124            25.24.23.22.21.19.18.17             1125            25.24.23.22.21.20.19.18.17             1126            25.24.23.22.21.20.19.18.17             1127            25.24.23.22.21.19.18.17             1128            25.24.23.22.21.19.18.17             1129            25.24.23.22.21.19.18.17             1130            25.24.23.22.21.19.18.17             1131            25.24.23.22.21.19             1132            25.24.23.22             1133            25.24.23             1134            25.24.23             1135            24.23             1136            24.23     7       1685            25.24.23.22.21.19.18             1686            25.24.23.22.21.19.18.17             1687            25.24.23.22.21.19.18.17             1688            25.24.23.22.21.20.19.18.17             1689            25.24.23.22.21.18             1690            25.24.23.22.21.19.18.17             1691            25.24.23.22.21.20.19.18.17             1692            25.24.23.22.21.19.18.17             1693            25.24.23.22.19.15.17             1694            25.24.23.22.19.18.17             1695            25.24.23.22.18.17             1696            24.23.22             1697            24.23     8       5932            17             5995            22.21             5996            21.20.19.18             5997            19.18.17             5998            18.17             5999            17             6004            21             6007            17      .sup. 8A             6277            23             6278            22,21             6279            21,20             6280            20     9       6548            24.23.22             6549            23.22             6550            22             6551            22             6560            21             6562            19             6563            18             6564            17     ______________________________________      *The numbers indicate the 26mers, 25mers, 24mers, etc., down to 17mers      that are contained within the oligo sequence shown. Size reduction in      individual oligo is limited to removal of nucleotides from the 5prime end      only of the oligo shown at each nucleotide starting position.

                  TABLE 5     ______________________________________     Preferred Size Variants of MRP-Oligos at different starting positions     in the MRP gene sequence hotspots     HOT-     Nucleotide     SPOTS    starting position                              Oligo Size Variants     ______________________________________     1         20             17               21             17               22             17               24             26               36,39,42,45,48,51,54                              25.24.23.22.21.19.18.17               37,40,43,46,49,52                              25.24.23.22.21.19.18.17               38,41,44,47,50,53                              25.24.23.22.21.19.18.17               55             25               56             25               57             25     2         169            26.19.18.17               170            18.17               171            17     3         219            19               220            25.24.23.22.21.20.19               221            24.23.22.21.19               222            23.22.21.20.19.18.17               223            22.21.20.19.18.17               224            21.19.18               225            20.19.18               226            19.18               227            18               236            19               263            25.24               264            25.24     4        1198            24              1199            24.23              1200            24.23              1201            24.23              1202            25.24.23.22              1203            24.23.22              1204            25.24.23.22              1205            25.24.23.22.21              1206            25.24.23.22.21              1207            25.24.23.22.21              1208            25.24.23.22.21              1209            25.23.22.21              1210            25.22.21.20.19.17              1211            25.21.17              1212            25.21.17              1213            24.21.18.17              1214            25.17              1215            25.17              1216            25.17              1217            25.17     5        2101            25.24.23.21.20.19.18.17              2102            25.24.23.21.20.19.18.17              2103            25.24.23.21.20.19.18.17              2104            25.24.23.21.20.19.18.17              2105            25.24.23.21.20.19.18.17              2106            25.24.23.21.20.19.18.17              2107            25.24.23.22.21.20.19.18.17              2108            25.24.23.22.21.20.19.18.17              2109            24.23.22.21.20.19.18.17              2110            23.22.21.20.19.18.17              2111            22.21.19.18              2112            22.21.19              2113            21              2114            20              2115            19     6        2469            25.24.23              2470            25.24.23              2471            25.24.23              2472            25.24.23.22              2473            25.24.23.22.21              2474            25.24.23.22.21              2475            25.24.23.22.21              2476            24.23.22.21              2477            23.22.21              2478            22.21              2479            21              2489            22.21              2490            21              2516            20.17              2517            17              2518            19.18.17              2519            20.19.18.17              2520            18.17              2521            17     7        2805            25              2829            25.24.23.22.21              2830            24.23.22.21              2831            23.22.21              2832            22.21.19              2833            21.19              2834            20.19              2835            19.18              2836            18              2837            18.17              2848            20.19.18.17              2849            19.18              2850            18              2862            24.23.22.21.19              2863            25.24.23.22              2864            25.24.23.22              2865            25.24.23.22              2866            24.23.22              2867            24.23              2868            24.23              2869            24.23              2870            25.24.23              2871            25.24.23              2872            24.23              2873            23     8        3528            25.24.23.22.21.20.19.18.17              3529            25.24.23.22.21.20.19.18.17              3530            25.24.23.22.21.20.19.18.17              3531            25.24.23.22.21.20.19.18.17              3532            25.24.23.22.21.20.19.18.17              3533            25.24.23.22.21.20.19.18.17              3534            25.24.23.22.21.19.18              3535            25.24.23.22.21.19.18              3536            25.24.23.22.21.19.18              3537            25.24.23.21.19              3538            25.24.23.22.21.20.19.18              3539            25.24.23.22.21.20.19.18              3540            25.24.23.22.21.20.19.18              3541            24.23.22.21.20.19.18              3542            23.22.21.20.19.18              3543            22.21.20.19.18              3544            21.19.18              3545            21.19              3546            19              3547            19.18              3548            18     9        4146            25.24.23.22              4147            24.23.22              4148            23.22.21              4149            22.21.19              4150            21.19              4151            25.24.23.22.21.19.18              4152            25.24.23.22.21.19.18              4153            25.24.23.22.21.19.18              4154            25.24.23.22.21.20.19              4155            25.24.23.22.19              4156            25.24.23.22              4157            25.24.23.22.21              4158            25.24.23.22.21.19              4159            24.23.22.21.19              4160            23.22.21.20.19.18              4161            22.21.20.19.18              4162            21.20.19.18              4163            20.19.18.17              4164            19.18.17              4165            19.18.17              4166            18.17              4167            17              4173            17     10       4873            25              4929            25.24.23.22              4930            25.24.23.22.21              4931            25.24.23.22.21.19              4932            25.24.23.22.21.19              4933            24.23.22.21.20.19.18              4934            23.22.21.19.18              4935            22.21.19.18              4936            21.20.19.18              4937            20.19.18              4938            19.18              4939            18              4940            17              4633            25,24,23,22,21              4634            24,23              4635            23              4636            22,21,20,19,18              4637            21              4639            19              4640            19,18     ______________________________________

Use of Selected Oligonucleotides for Systemic Treatment of Patients

Pursuant to the invention, oligos designed to inhibit the expression of the MDR1 or MRP genes are administered to patients in accordance with any of a number of standard routes of drug administration. For example, the oligo may be administered to a patient by continuous intravenous administration for a period of time such as ten days. An infusion rate of 0.05-0.5 mg/kg/hr should be suitable for the practice of the invention. Alternatively, the oligo may be injected daily, given orally or be released into the patient's body from an implanted depot or be given by some other route of administration as deemed appropriate according to the criteria of standard clinical practice.

Other inhibitors of P-gp or MRP function may be given in conjunction with the administration of the oligonucleotide in accordance with the best mode of use for the given agent. If a patient has cancer or a premalignant syndrome and the purpose of administering the oligo to the patient is to improve chemotherapy response, then chemotherapeutic agents will be given either during or at about the same time as the oligo administration. A period of about one week prior to or following the administration of the oligo should be a period of time during which chemotherapeutic drugs should be given to the patient. If the patient has cancer or a premalignant syndrome and the purpose of administering the oligo is to prevent the development of multidrug resistance in the diseased cells, then the oligos will be administered to patients at times when the patient does not have active levels of chemotherapeutic drugs in the patient's body. If a patient has an autoimmune disease, such as arthritis, is experiencing graft rejection or graft-versus-host disease, then the oligos may be administered to the patient alone or in combination with other agents including inhibitors of P-gp or MRP as an immunosuppressive therapy. Oligo doses and schedules suitable for cancer patients also should be suitable for patients with autoimmune disease or who are experiencing graft rejection. Drugs cytotoxic or cytostatic to cells of the patient's immune system may also be given in conjunction with the oligo to bring about an immunosuppressed state in the patient for the purpose of reversing the pathological conditions.

Use of Selected oligonucleotides for Depleting Malignant Cells or Cytotoxic Mononuclear Cells From Bone Marrow or From Peripheral Stem Cell Harvests

Pursuant to the invention, one first obtains a sample of bone marrow or peripheral stem cells in accordance with any of a number of standard techniques. The patient to receive an autologous transplant may then be treated with an optimal dose of radiation and/or chemotherapy according to standard clinical procedures.

The bone marrow or peripheral stem cell sample may be cryopreserved and stored until needed, or immediately treated with the oligo.

In order for the tumor cell or normal mononuclear cell targets to be affected by the oligo, the cells must be exposed to the oligos under conditions that facilitate their uptake by the malignant cells. This may be accomplished by a number of procedures, including, for example, simple incubation of the cells with an optimal concentration of the oligo in a suitable nutrient medium for a period of time suitable to achieve a significant reduction in P-gp or MRP expression. Four days should be sufficient incubation period, but time may need to be extended, e.g., for slow growing tumors. At this time, a chemotherapeutic drug may be added that will kill any cancer cells present in the graft in the case of an autologous transplant or non-malignant mononuclear cells that can produce graft-versus-host disease in the case of an allogeneic transplant. After the bone marrow or peripheral stem cells have been cultured as just described, they are then infused into the transplant recipient to restore hemopoiesis.

Aptameric Oligonucleotides Capable of Reversing Multidrug Resistance Phenotype

The MDR-aptameric oligonucleotides shown in Table 14 (Example 7) are very active in reversing the multidrug resistance phenotype, and in inhibiting the growth of multidrug resistant cancer cells. They have little or no drug sensitizing or proliferation-inhibiting activity on drug sensitive cells. This aptameric effect also is shared by the OL(1C)mdr oligo and, to a lesser extent, by the OL(1Q)mdr and SJ(34)mdr oligos. It has been found, using a previously described technique involving purified rat brain protein kinase C isoenzymes (Ward et al., J. Biol. Chem . 270: 8056, 1995) that these aptameric oligonucleotides do not significantly alter the activity of PKC-α, -β or -gamma. The existing data support the concept of developing these aptamers for clinical use, using basically the same strategy described herein for oligonucleotides targeting MDR1 or MRP. Using methodology of the type described by Schultze et al (J. Mol. Biol. 235: 1532, 1994), common structural features in these aptamers can be uncovered that would provide a basis for designing additional (and perhaps more specific and more active) aptamers for reversing multidrug resistance. In addition, using standard molecular biological techniques such as those described in standard texts such as, for example, Current Protocols in Molecular Biology (FM Ausubel et al., eds, New York: John Wiley & Sons, Inc., 1994), these MDR-aptameric oligonucleotides can be used to identify the target molecule to which they bind and produce the effect seen on multidrug resistance.

For example, these MDR-aptamers can be radiolabeled and incubated in vitro with cells which are then lysed; or, the MDR-aptamers can be used to treat cell lysates, and fractionation studies can then be carried out to isolate molecules to which the radiolabeled MDR-aptamer binds. Studies would then be carried out to confirm that a particular molecule to which the MDR-aptamer had been tightly bound has a functional role in multidrug resistance. For example, it might be shown that the molecule to which the MDR-aptamer binds is more active, or is expressed at higher levels, in multidrug resistant cells than in the drug-sensitive counterparts.

Purification of the protein to which the aptamers bind would allow microsequencing. The protein sequence can be used to generate a set of oligonucleotide probes that can be used to identify the cDNA and/or gene sequence that encodes the said protein. If it is a known protein or gene, then this information will identify the target of the MDR-aptamers. If it is not a known gene, then the gene can be cloned and characterized. Antisense oligos against this gene would be expected to have activity in reversing multidrug resistance. Even if the gene encoding the protein to which the MDR-aptamers bind is not novel, then observation that targeting this protein with aptameric oligos leads to a reversal of the multidrug resistance phenotype is a novel observation, and it provides the basis for a new therapeutic strategy for the treatment of cancer.

In sum, preferred embodiments of the present invention relate to the systemic administration of an oligonucleotide capable of inhibiting the expression of one or more of these pumps in the tumors of patients with cancer; or administering two oligos, one capable of inhibiting, for example, P-gp expression and the other inhibiting, for example, MRP expression, thereby rendering the patient's tumor more susceptible to the cytotoxic effects of chemotherapeutic agents administered together with the oligo. Also, these oligos may be administered to patients prophylactically to prevent the development of multidrug resistance in a tumor. These oligos may be used alone to inhibit multidrug resistance, or in combination with other inhibitors of P-gp or MRP. The invention also includes procedures and compositions for ex vivo administration to purge malignant cells from bone marrow grafts. Said oligos may also be administered to transplant or autoimmune patients as immunosuppressive agents.

In another application, these oligos may be used to interrupt the blood brain barrier, thus allowing therapeutic agents to pass from the general circulation into the central nervous system.

In yet another application, MRP oligos may be of use in inhibiting the transport of leukotrienes across cell membranes, and may be of clinical use in situations where this class of compounds is involved, for example, in inflammatory/allergic responses.

Also revealed are prototype oligos that can inhibit various multidrug resistance phenotypes in cancer cells by an aptameric effect.

The following examples are provided to describe the invention in further detail. These examples are intended to illustrate, and not to limit, the invention.

EXAMPLE 1 Sensitization of Multidrug-Resistant (MDR+) Cancer Cell Lines to Doxorubicin by MDR-Oligos

Multidrug resistant tumor cells were incubated with 15μM of OL(6)mdr oligo, Cohen(1)mdr oligo or a negative-control (HIV-2) oligo for 4 days at 37° C. in a humidified incubation chamber. Varying doses (5-fold serial dilutions) of doxorubicin (from 1×10⁻⁵ M to 1×10⁻⁸ M) were then added to the culture wells after the cells had been subcultured into media without oligos; cells were then incubated in the same culture environment for an additional three days. Radiolabeled (tritiated) thymidine was added to all cultures for 24 hr before harvest. Each data point is the mean of 4 replicates. The data (Table 6) demonstrate that OL(6)mdr is able to sensitize multidrug-resistant cancer cells to chemotherapeutic agents, nearly three times better than the best MDR-oligo reported in the literature (Jarozewski et al.: Cancer Comm. 2: 287-294, 1990.).

                  TABLE 6     ______________________________________     Sensitization of 8226/Dox cells to killing by doxorubicin following     incubation with OL(6)mdr MDR-oligo.                                       Relative                  SEQ       IC.sub.50  Sensitization     Treatment    ID NO.    dose (× 10-.sup.7 M)                                       (fold increase)     ______________________________________     Medium only  --        6.5                  --     HIV-2 control oligo*                  85        7.0                  --     Cohen(1)mdr oligo**                  86        5.0                  1.3     OL(6)mdr oligo                  1         1.8                  3.6     ______________________________________      *HIV-2 nucleotide sequence (20mer): 5TGTCTCCGCT TCTTCCTGCC3' (SEQ ID NO:      85);      **Cohen(1)mdr nucleotide sequence (15mer): 5GCTCCTCCAT TGCGG3' (SEQ ID NO      86)

EXAMPLE 2 Sensitization of Multidrug-Resistant 8226/Dox Human Myeloma Cells and CEM/VBL10 Human Leukemia Cells to Vincristine Following Incubation with MDR-Oligos Under Low Oxygen Levels

As shown in this example, several oligos which target the MDR1 gene are able to sensitize multidrug-resistant cell lines to the cytotoxic effects of the chemotherapeutic agent Vincristine. Tumor cells were treated in vitro with 10 μM of the indicated oligo for 4 days at 37° C., then counted, dispensed into individual tubes, and pulsed with Vincristine at various doses (serial 5-fold dilutions) for 3 hrs. The cells were then washed and seeded into 96-well plates (quadruplicate replicates) for pulsing with tritiated thymidine 3 days later. Cells were harvested the next day (=4th day after drug treatment). Tritiated thymidine uptake was analyzed by liquid scintillation.

The Prototype MDR-oligos shown in Tables 1 and 2 represent the most potent of the MDR-oligos screened on the tumor cell line in these studies. In experiments not shown, most of the other phosphorothioate MDR-oligos evaluated in vitro (sequences not listed) had little-to-no capacity to sensitize targeted tumor cells to cytotoxic drugs at concentrations at which the prototype oligos had substantial activity, confirming that the dramatic antisense effects noted above were not merely a cellular response to exposure to phosphorothioate molecules, but appear to be sequence dependent.

The OL(1)mdr oligo (Table 7) is clearly much more potent than OL(6)mdr in sensitizing 8226/Dox cells to chemotherapeutic agents. Given the data in Tables 6 and 7, it is also evident that OL(1)mdr is much more potent than the Cohen(1)mdr oligo, even though the OL(1)mdr and Cohen(1)mdr oligos have overlapping sequences:

OL(1)mdr 5'-GCTCCTCCAT TGCGGTCCCC-3'

(SEQ ID NO. 25)

Cohen(1)mdr 5'-GCTCCTCCAT TGCGG-3'

(SEQ ID NO. 86)

The present inventor, therefore, has made the unobvious discovery that the inclusion of the sequence ("TCCCC") to the oligos that bind in the same general area as the Cohen(1)mdr oligo greatly increases the potency of the oligos in terns of sensitizing cells that express MDR1 to chemotherapeutic agents.

                  TABLE 7     ______________________________________     Sensitization or multidrug-resistant tumor cells to killing by     Vincristine     following incubation with several MDR-oligos in 5% oxygen                                         Relative             Oligo              IC.sub.50 dose                                         sensitization     Cell Type             Used     SEQ ID NO.                                (× 10-.sup.7 M)*                                         (fold increase)     ______________________________________     8226/Dox             Control  --        4.5      --             OL(1)mdr 25        0.027    167             PA(1)mdr 40        0.28     16             5(1)mdr  10        1.80     2.5             OL(6)mdr 1         2.20     2.5     CEM/    Control  --        100.0    --     VLB10   5(1)mdr  10        0.9      111             OL(6)mdr 1         1.2      83             OL(5)mdr 21        1.5      67             OL(1)mdr 25        1.6      62             SJ(36)mdr                      39        1.6      62             OL(2)mdr 35        1.6      62             PA(1)mdr 40        1.9      53             OL(3)mdr 37        2.0      50     ______________________________________      *IC.sub.50 = inhibitory concentration which gives 50% reduction in      tritiated thymidine uptake into DNA

EXAMPLE 3

As shown in this example, it was determined that there was an increase in sensitivity of multidrug-resistant 8226/Dox human myeloma cells to Vincristine with increasing time of incubation following initial exposure to Vincristine. The experimental conditions were identical to those ustilized in obtaining the data shown in Table 7, except that the present experiment had two additional time points.

                  TABLE 8     ______________________________________     Increase in sensitivity of multidrug-resistant 8226/Dox human     myeloma cells to Vincristine with increasing time of incubation     following initial exposure to Vincristine                SEQ    Oligo      Relative Sensitization     Oligo Used ID     Concentration                                  (fold increase)     (Trivial Name)                NO.    (μM)    Day 5*                                        Day 7 Day 9     ______________________________________     5(1)mdr    10     2          3.4   26.9  724                       10         2.1   20.5  423     OL(1)mdr   25     2          18.3  412   5500                       10         75.8  389   5729     PA(1)mdr   40     2          2.3   27    1309                       10         6.3   28    550     OL(6)mdr   1      2          1.6   3.5   61                       10         1.8   15.9  550     Control Oligo**                87     2          1.0   0.9   0                       10         0.8   7.0   26.2              IC.sub.50 Value on:                Day 5       Day 7    Day 9     Media Control                4.4 × 10.sup.-7 M                            7 × 10.sup.-6 M                                     5.5 × 10.sup.-4 M     ______________________________________      *Days after initial treatment exposure with Vincristine      **Control oligo sequence: 5CCTCGGTCCC CCCTCGTCCC C3' (SEQ ID NO. 87)

EXAMPLE 4 In Vitro Sensitization of MRP-Expressing Lung Cancer Cells to Etoposide by MRP-Oligos

The Prototype MRP-ODN shown in Table 3 represent the most potent of the MRP-ODNs screened on the tumor cell line in these studies. The human non-small-cell lung adenocarcinoma cell line A427 was treated with oligos at various concentrations for 4 days in vitro. The cells then were pulsed with various concentrations of etoposide (range 1.6×10⁻⁴ to 1×10⁻⁶ M) for 3 hrs. Next, cells were trypsinized and seeded by volume into 96-well plates with 12 replicates per treatment condition. Three days later, tritiated thymidine was added and the cells harvested the next day.

The OL(8)MRP antisense oligo specific to an MRP gene-related sequence made the A427 lung cancer cell line 18-times more sensitive in vitro to the cytotoxic effects of etoposide (VP-16). The data (Table 9) confirm the specificity of this activity. OL(6)mdr, which targets transcripts of the MDR1 gene, was used as a negative control with this MRP-expressing cell line. When novobiocin, a putative MRP inhibitor, was used at the same time as VP-16, it increased the A427 drug sensitivity by about 5 fold.

                  TABLE 9     ______________________________________     A427 Lung Cancer Cell Line Treated with MRP-oligos             SEQ  Oligo  Etoposide (VP-16)               ID     Level           Relative Sensitization     Treatment NO.    (μM)                             IC.sub.50                                      (fold increase)     ______________________________________     Experiment 1     Media-Control               --     --     3.6 × 10.sup.-5 M*                                      --     Novobiocin               --     --     0.72 × 10.sup.-5 M                                      5.0     OL(6)mdr control                1     10.0   2.2 × 10.sup.-5 M                                      1.6     OL(6)mdr control                1     5.0    3.0 × 10.sup.-5 M                                      1.2     OL(6)mdr control                1     2.5    4.8 × 10.sup.-5 M                                      0     OL(8)MRP  64     10.0     2 × 10.sup.-6 M                                      18.0     OL(8)MRP  64     5.0    1.0 × 10.sup.-5 M                                      3.6     OL(8)MRP  64     2.5    1.8 × 10.sup.-5 M                                      2.0     Experiment 2     Media Control               --     --       5 × 10.sup.-5 M                                      --     OL(6)MRP  60     10.0     6 × 10.sup.-6 M                                      8     5(2)MRP   43     10.0     8 × 10.sup.-6 M                                      6     OL(3)MRP  62     10.0     9 × 10.sup.-6 M                                      5     5(3)MRP   42     10.0     1 × 10.sup.-5 M                                      5     Experiment 3     Media Control               --     --     6.0 × 10.sup.-5 M                                      --     OL(5)MRP  48     10     1.0 × 10.sup.-5 M                                      6     OL(14)MRP 44     10     1.9 × 10.sup.-5 M                                      3     OL(2)MRP  49     10     2.5 × 10.sup.-5 M                                      2.4     Experiment 4     OL(6)mdr control                1     1      5.0 × 10.sup.-5 M                                      0     OL(4)MRP  76     1      1.6 × 10.sup.-5 M                                      3     ______________________________________      *IC.sub.50 = inhibitory concentration which gives 50% reduction in      tritiated thymidine uptake

EXAMPLE 5

In this example, experimental conditions were identical to those utilized in obtaining the data in Example 2. These data show that the MRP-oligo, OL(8)MRP, can substantially sensitize non-lung cancer cells to chemotherapeutic agents, as determined in in vitro assays.

                  TABLE 10     ______________________________________     In vitro sensitization of 8226/Dox cells to Vincristine by an MRP-oligo     and comparison to several MDR-ODNs for relative potency.                         IC.sub.50  Relative Sensitization     Treatment SEQ ID NO.                         dose (× 10-.sup.7 M)                                    (fold increase)     ______________________________________     Medium only               --        11         --     OL(8)MRP  60        0.55       20     OL(1)mdr  25        0.2        55     OL(6A)mdr 2         0.88       12.5     OL(3)mdr  37        1.0        11     OL(2)mdr  35        3.0        3.7     ______________________________________

EXAMPLE 6

In vitro testing of MDR-Oligonucleotides was done on the multidrug-resistant (MDR+) RPMI-8226/Dox4 human multiple myeloma cell line (gift of Dr. William Dalton, Univ. Arizona Cancer Ctr., Tucson). Cells were incubated in vitro for 4 days at 37° C. with MDR-oligos at 0.2 μM final concentration; pulsed 18 hr with serial 5-fold dilutions of chemotherapeutic drug (vincristine) from 1×10⁻⁴ M to 2×10⁻¹⁰ M. Cells were then washed, and incubated for an additional 4 days. Tritiated thymidine (³ H-TdR) was added for last 18 hr to measure status of cellular proliferation. Controls included MDR+8226 cells similarly treated with vincristine but pretreated either with (a) control ODNs, or (b) culture medium only.

                  TABLE 11a     ______________________________________     MDR--ODNs                             Oligo              SEQ    5'-end  length                                   Why the site     Oligo    ID     target  (No. of                                   was selected                                             Relative     Name     No.    site.sup.1                             bases)                                   (Footnote #)                                             Activity     ______________________________________     OL(1)mdr 25     1125.sup.1                             20              +++     OL(1A)mdr              34     1122    20    variant   ++     OL(1B)mdr              26     1123    22    variant   ++++++     OL(1C)mdr              27     1125    25    variant   +++++++     OL(1Q)mdr              28     1125    23    variant   ++++++     OL(1W)mdr              29     1125    18    variant   +++++     OL(1Wa)mdr              30     1123    18    variant   ++++     OL(1Wb)mdr              31     1125    16    variant   ++++     OL(1Wc)mdr              32     1121    18    variant   ++++     OL(1X)mdr              33     1127    18    variant   +++     OL(2)mdr 35     1688    20              +     OL(3)mdr 37     5996    20              +     OL(5)mdr 21     1000    20              +++     OL(6)mdr 1      488     20              +     OL(6A)mdr              2      496     20    variant   --     OL(7)mdr 110    2199    20              +     OL(8)mdr 111    5722    20              --     OL(9)mdr 112    3881    20              --     OL(10)mdr              11     688     20              +++     OL(11)mdr       851     20              +     OL(12)mdr              12     884     20              +++     OL(12A)mdr              13     881     22    variant   +++++     OL(12B)mdr              14     885     18    variant   +++     OL(12C)mdr              15     881     18    variant   ++     OL(13)mdr       958     20              --     OL(14)mdr       5713    20              --     OL(15)mdr              16     941     20              --     OL(16)mdr              3      517     20              +     SJ(1)mdr 113    85      20    splice junction                                             --     SJ(2)mdr 114    673     20    splice junction                                             ++     SJ(6)mdr        2559    20    splice junction                                             +     SJ(18)mdr       6074    20    splice junction                                             +     SJ(30)mdr       4867    20    splice junction                                             --     SJ(33)mdr       349     20    splice junction                                             --     SJ(34)mdr              5      540     20    splice junction                                             +++     SJ(34A)mdr              6      542     18    variant   +++++     SJ(34B)mdr              7      540     22    variant   ++     SJ(34C)mdr              8      533     20    varianl   +     SJ(34D)mdr              9      543     16    variant   +     SJ(35)mdr       1097    20    splice junction                                             --     SJ(36)mdr              39     6551    22    splice junction                                             +     3(1)mdr         7051    20    3'-end    --     5(1)mdr  10     664     26    2, AUG start                                             ++     5(2)mdr         640     28    2         --     AP(1)mdr 41     670     23    3; TR binding                                             +     AP(4)mdr        636     22    3; TR binding                                             +     PA(1)mdr 40     --      23    reverse of AP(1)                                             +     TH(2)mdr        2954    20    published +     CAP(2)mdr       556     22    cap site  +     LOW(3)mdr       11      20    low Tm    +     Cohen(1)mdr              86     1130    15    published +     NF-kB(1)mdr     296     22    3; TR binding                                             --     CAT(L)mdr       432     20    TR binding                                             --     Y-box-mdr       464     22    TR binding                                             --     ______________________________________      Reactivity: -- = no effect; + = weak positive effect; +++++++ = very      strong positve effect.      The relative reactivity indicated for each MDR--ODN summarizes results      obtained with 8226/Dox4, 8226/Dox6 and CEM/VLB10 multidrugresistant cell      lines.      .sup.1 The numbering for the 5end target site is based on Genbank entry      HUMMDR1A01through-HUMMDR1A26 (Chin et. al., Mol. Cell. Biol. 9: 3808,      1989; Chen et al., J. Biol. Chem. 265: 506, 1990), considered as a      continuous sequence with the nucleotide at the extreme 5end of the      sequence being given the nucleotide base number 1.      .sup.2 These ODNs were designed to have sufficient binding affinity to th      5untranslated portion of the cDNA to potentially block the movement of th      ribosome toward the AUG start site.      .sup.3 Binding sites of these oligos are within an enhancer for the MDR1      gene, the sequence of which is reported by Kohno et al, J. Biol. Chem.      265: 19690, 1990. (GenBank entry # HUMMDR1B/J05674).      .sup.4 OL(7)mdr Sequence ID No. 110 TAGCCACATGGCCCCAGGAA      OL(8)mdr Sequence ID No. 111 ACTGACTTGCCCCACGGCCA      OL(9)mdr Sequence ID No. 112 CCAAAGGGCAAAGGGCAAGG      SJ(1)mdr Sequence ID No. 113 GTACCTTACCTTTTATCTGG      SJ(2)mdr Sequence ID No. 114 TGCCCCTACCTCGCGCTCCT

                  TABLE 11b     ______________________________________     MRP-ODNs                               ODN                       5'-end  length                                     Why the site     Oligo     SEQ     target  (No. of                                     was selected                                              Relative     Name      ID NO.  site.sup.1                               bases)                                     (Footnote #)                                              Activity     ______________________________________     A(1)MRP           194.sup.1                               20    AUG start site                                              -     OL(2)MRP  49      2114    20             -     OL(3)MRP  62      2848    20             +++     OL(4)MRP  76      4154    20             -     OL(5)MRP  48      1210    20             +     OL(6)MRP  60      2516    20             +     OL(7)MRP          3155    20             -     OL(8)MRP  64      3539    20             +++++     OL(9)MRP          3800    20             +     OL(10)MRP         4484    20             ++     OL(11)MRP         4715    20             -     OL(12)MRP         89      20             -     OL(13)MRP         129     20             -     OL(14)MRP 44      220     20             +     OL(15)MRP 84      3312    20             -     OL(16)MRP         1580    20             -     3(2)MRP           4836    20    3'-end   -     3(3)MRP   81      4933    20    3'-end   -     5(2)MRP   43      164     26    2        +++     5(3)MRP   42      24      26    2        ++     LOW(1)MRP         351     20    low T.sub.m                                              -     LOW(2)MRP         714     20    low T.sub.m                                              +     CAP(2)MRP         1       19    Cap site +     ______________________________________      Reactivity: - = no effect; + = weak positive effect; +++++ = strong      positive effect      .sup.1 The numbering for the 5end target site is based on Genbank entry      HUMMRPX/L05628 (Cole et al., Science 258: 1650, 1992) with the nucleotide      at the extreme 5end of the sequence being given the nucleotide base numbe      1.      .sup.2 These ODNs were designed to have sufficient binding affinity to th      5untranslated portion of the cDNA to potentially block the movement of th      ribosome toward the AUG start site.

                                      TABLE 12     __________________________________________________________________________     IC.sub.50 SUMMARY     (based on 8226/Dox4 human myeloma cells)                                    Fold-Increase in     Oligos*       Sequence                        Oligo                            Vincristine                                    sensitivity     (trivial name)             SEQ ID NO.                   Position                        Length                            IC.sub.50                                    to drug treatment**     __________________________________________________________________________     Media Control             --    --   --  1.2 × 10.sup.-5 M                                    --     OL(1)mdr             25    1125 20  1.4 × 10.sup.-7 M                                    86     OL(1B)mdr             26    1123 22  1.6 × 10.sup.-9 M                                    7500     OL(1C)mdr             27    1125 25  <<1.6 × 10.sup.-9 M                                    >>7500     OL(1Q)mdr             28    1125 23  <1.6 × 10.sup.-9 M                                    >7500     OL(1W)mdr             29    1125 18  2.1 × 10.sup.-9 M                                    5714     OL(10)mdr             11    688  20  3.0 × 10.sup.-7 M                                    40     OL(12)mdr             12    884  20  6.0 × 10.sup.-8 M                                    200     OL(12A)mdr             13    881  22  3.2 × 10.sup.-8 M                                    375     OL(12B)mdr             14    885  18  3.0 × 10.sup.-7 M                                    40     SJ(34)mdr             5     540  20  4.1 × 10.sup.-8 M                                    293     SJ(34A)mdr             6     542  18  <1.6 × 10.sup.-9 M                                    >7500     SJ(34C)mdr             8     533  20  5.5 × 10.sup.-9 M                                    2182     __________________________________________________________________________      *All oligonucleotides were tested at a final concentration of 0.2 μM      **Fold increase in drug sensitivity compared to media control (no oligo o      drug).

EXAMPLE 7 Mathematical/Statistical Model Used to Analyze ODN- and Drug-Testing Data

The following mathematical/statistical model was used to analyze the effects of oligos and various drug treatments on targeted tumor cells in these studies. The model is based on experiments which generally involve treatment of tumor cells with one of several ODNs or media only, and with several dose levels of an anti-cancer agent (such as, for example, vincristine (VCR)) or media only. Analysis goals were to model the relationship of cell kill to the dose of VCR (for example), and to compare the effects of the various ODNs.

Investigation of the data indicate that the logarithm of counts can be modelled as a function of the dose of drug for each ODN; i.e.:

    ln(count)=f(dose)+error

A "full model" that fits for each ODN and drug dose in a given experiment is as follows:

    ln(count)=α+β.sub.1 (drug)+β2(dose) +β.sub.3 (square root of dose) +β.sub.4 (fourth root of dose)+error

Thus, the natural log (count) is modelled as a linear function of powers of drug dose in the assay. For a fixed ODN, i, the expected count for media-only (no drug) is

    E(count).sub.media =exp(α.sub.i)

For the drug dose, d*, on the other hand, the expected count is

    E(count)d*=exp(α.sub.i +β.sub.2i d*+β.sub.3i  square root of d*!+β.sub.4i {fourth root of d*}).

Using standard linear regression techniques, the "full" model (19 regressor variables) was fitted to the data from all serial-dilution drug dose levels together; from this, a more parsimonious model was developed by removing regressor variables which did not contribute to the model fit. This final model contained 10 regressor variables.

From this most parsimonious model, with its 10 predictor variables, graphs of the data are prepared, in which Predicted ³ H-TdR uptake Counts (on the Y-axis) are plotted against Log₁₀ (dose of drug) (X-axis); this most parsimonious model fits the data with an excellent correlation coefficient of R² =0.98. The estimated functions (curved lines) associating Log₁₀ (Vincristine dose) with expected "³ H-TdR Counts" for each oligo can be calculated, and are shown in the following FIGURE, where the "Media only" control counts are placed on the left-hand Y-axis.

Various estimates of parameters from the parsimonious model are shown in TABLE 13, which contains the model estimate of the count for each ODN with media only; differences here (when compared to "media only" no ODN!) reflect the cell kill associated with the ODN alone. TABLE 13 also contains an estimate of IC₅₀ values, the dose of vincristine (VCR) which is estimated to produce an expected count which is exactly half that expected with "media only" no VCR!. While formal statistical comparisons of the IC₅₀ values of the various ODNs is not possible, an ordering of the ODNs by estimated IC₅₀ values is possible.

                                      TABLE 13     __________________________________________________________________________     Estimated Mean Count at VCR Dose = 0 and IC.sub.50 for Oligonucleotides     used to     treat 8226/Dox4 cells: Results from most parsimonious model     Oligonucleotide                    Estimated Mean  Fold Increase in     (trivial name)              SEQ ID NO.                    Count (cpm)                            Estimated IC.sub.50                                    drug sensitivity**     __________________________________________________________________________     Medla oniy              --    159,500 1.28 × 10 - 5M                                    --     OL(12)mdr              12    159,500 5.50 × 10 - 8M                                    233     OL(12B)mdr              14    159,500 7.39 × 10 - 7M                                    17.3     OL(12A)mdr              13    159,500 2.97 × 10 - 8M                                    414     OL(1B)mdr              26    120,800 1.79 × 10 - 8M                                    715     OL(1C)mdr              27     40,200 1.33 × 10 - 7M                                    96.2     OL(1Q)mdr              28     76,200 1.92 × 10 - 8M                                    667     SJ(34A)mdr              6     100,300 6.81 × 10 - 8M                                    188     __________________________________________________________________________      **fold increase in drug sensitivity compared to corresponding oligo      control (no drug)

                                      TABLE 14     __________________________________________________________________________     Sensitization of 8226/Dox Cells to Killing by VCR     Following Incubation with MDR-aptameric oligonucleotides           SEQ ID               Sequence                Fold Increase in     Treatment           No. (5'-->3')         IC.sub.50                                       Drug Sensitization**     __________________________________________________________________________     Media --  --                4 × 10.sup.-5 M                                       --     MDR-APT-1           88  CCCCTGGCGT TACCTCCTCG TTTCT                                 1 × 10.sup.-9 M                                       40,000     MDR-APT-2           89  TTCGCCTGAT TTCCGCCTCC CGTCT                                 2 × 10.sup.-9 M                                       20,000     MDR-APT-3           90  CGGTCCGTTA TGTTCCTG                                 7 × 10.sup.-9 M                                       5,714     MDR-APT-4           91  ACTCGCCTCC CACGTAGTGC TT                                 <1 × 10.sup.-9 M                                       >40,000     __________________________________________________________________________      **Fold increase in drug sensitivity compared to media control

It will be appreciated that changes may be made in the nature, composition, operation and arrangement of the various elements described herein without departing from the spirit and scope of the invention as set forth in the following claims.

    __________________________________________________________________________     SEQUENCE LISTING     (1) GENERAL INFORMATION:     (iii) NUMBER OF SEQUENCES: 114     (2) INFORMATION FOR SEQ ID NO:1:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:     CCCACGCCCCGGCGCTGTTC20     (2) INFORMATION FOR SEQ ID NO:2:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:     GTGCTCAGCCCACGCCCCGG20     (2) INFORMATION FOR SEQ ID NO:3:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:     GGCAAAGAGAGCGAAGCGGC20     (2) INFORMATION FOR SEQ ID NO:4:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:     TGGCAAAGAGAGCGAAGCGG20     (2) INFORMATION FOR SEQ ID NO:5:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:     TCGAATGAGCTCAGGCTTCC20     (2) INFORMATION FOR SEQ ID NO:6:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 18 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:     TCGAATGAGCTCAGGCTT18     (2) INFORMATION FOR SEQ ID NO:7:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 22 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:     ACTCGAATGAGCTCAGGCTTCC22     (2) INFORMATION FOR SEQ ID NO:8:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:     AGCTCAGGCTTCCTGTGGCA20     (2) INFORMATION FOR SEQ ID NO:9:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 16 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:     CGAATGAGCTCAGGCT16     (2) INFORMATION FOR SEQ ID NO:10:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 26 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:     CCCTACCTCGCGCTCCTTGGAACGGC26     (2) INFORMATION FOR SEQ ID NO:11:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:     GCTCCCAGCTTTGCGTGCCC20     (2) INFORMATION FOR SEQ ID NO:12:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:     GCGCGCTCCGGGCAACATGG20     (2) INFORMATION FOR SEQ ID NO:13:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 22 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:     CGCGCTCCGGGCAACATGGTCC22     (2) INFORMATION FOR SEQ ID NO:14:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 18 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:     CGCGCTCCGGGCAACATG18     (2) INFORMATION FOR SEQ ID NO:15:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 18 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:     CTCCGGGCAACATGGTCC18     (2) INFORMATION FOR SEQ ID NO:16:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:     TGCTTCCTCCCACCCACCGC20     (2) INFORMATION FOR SEQ ID NO:17:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:     TCCTCCCACCCACCGCCCGC20     (2) INFORMATION FOR SEQ ID NO:18:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:     TTCCTCCCACCCACCGCCCG20     (2) INFORMATION FOR SEQ ID NO:19:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:     CTTCCTCCCACCCACCGCCC20     (2) INFORMATION FOR SEQ ID NO:20:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:     GCTTCCTCCCACCCACCGCC20     (2) INFORMATION FOR SEQ ID NO:21:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:     TCTGGACTTTGCCCGCCGCC20     (2) INFORMATION FOR SEQ ID NO:22:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:     TTCTGGACTTTGCCCGCCGC20     (2) INFORMATION FOR SEQ ID NO:23:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:     GTTCTGGACTTTGCCCGCCG20     (2) INFORMATION FOR SEQ ID NO:24:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:     CGTTCTGGACTTTGCCCGCC20     (2) INFORMATION FOR SEQ ID NO:25:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:     GCTCCTCCATTGCGGTCCCC20     (2) INFORMATION FOR SEQ ID NO:26:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 22 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:     GCTCCTCCATTGCGGTCCCCTT22     (2) INFORMATION FOR SEQ ID NO:27:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 25 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:     TCTTTGCTCCTCCATTGCGGTCCCC25     (2) INFORMATION FOR SEQ ID NO:28:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 23 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:     TTTGCTCCTCCATTGCGGTCCCC23     (2) INFORMATION FOR SEQ ID NO:29:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 18 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:     TCCTCCATTGCGGTCCCC18     (2) INFORMATION FOR SEQ ID NO:30:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 18 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:     CTCCATTGCGGTCCCCTT18     (2) INFORMATION FOR SEQ ID NO:31:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 16 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:     CTCCATTGCGGTCCCC16     (2) INFORMATION FOR SEQ ID NO:32:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 18 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:     CCATTGCGGTCCCCTTCA18     (2) INFORMATION FOR SEQ ID NO:33:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 18 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:     GCTCCTCCATTGCGGTCC18     (2) INFORMATION FOR SEQ ID NO:34:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:     CCTCCATTGCGGTCCCCTTC20     (2) INFORMATION FOR SEQ ID NO:35:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:     GCAACCAGCACCCCAGCACC20     (2) INFORMATION FOR SEQ ID NO:36:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:     GCAGCAACCAGCACCCCAGC20     (2) INFORMATION FOR SEQ ID NO:37:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:     TGCCCACCAGAGCCAGCGTC20     (2) INFORMATION FOR SEQ ID NO:38:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 22 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:     GCCTCCTTTGCTGCCCTCACGA22     (2) INFORMATION FOR SEQ ID NO:39:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 22 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:     CCAGGGCTTCTTGGACAACCTA22     (2) INFORMATION FOR SEQ ID NO:40:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 23 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:     GCGGGAGGTGAGTCACTGTCTCC23     (2) INFORMATION FOR SEQ ID NO:41:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 23 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:     GGAGACAGTGACTCACCTCCCGC23     (2) INFORMATION FOR SEQ ID NO:42:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 26 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:     CGGCGGCGGCGGCGCAGGGAGCCGGG26     (2) INFORMATION FOR SEQ ID NO:43:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 26 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:     CGGTGGCGCGGGCGGCGGCGGGCACC26     (2) INFORMATION FOR SEQ ID NO:44:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:     GCGGGTCGGAGCCATCGGCG20     (2) INFORMATION FOR SEQ ID NO:45:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:     GAGCGGGTCGGAGCCATCGG20     (2) INFORMATION FOR SEQ ID NO:46:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:     AGAGCGGGTCGGAGCCATCG20     (2) INFORMATION FOR SEQ ID NO:47:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:     CCAGAGCGGGTCGGAGCCAT20     (2) INFORMATION FOR SEQ ID NO:48:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:     CTGCGGCCCGGAAAACATCA20     (2) INFORMATION FOR SEQ ID NO:49:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:     CGGTGATGCTGTTCGTGCCC20     (2) INFORMATION FOR SEQ ID NO:50:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:     CGTGCCCCCGCCGTCTTTGA20     (2) INFORMATION FOR SEQ ID NO:51:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:     TCGTGCCCCCGCCGTCTTTG20     (2) INFORMATION FOR SEQ ID NO:52:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:     TTCGTGCCCCCGCCGTCTTT20     (2) INFORMATION FOR SEQ ID NO:53:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:     GTTCGTGCCCCCGCCGTCTT20     (2) INFORMATION FOR SEQ ID NO:54:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:     TGTTCGTGCCCCCGCCGTCT20     (2) INFORMATION FOR SEQ ID NO:55:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:     CTGTTCGTGCCCCCGCCGTC20     (2) INFORMATION FOR SEQ ID NO:56:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:     GCTGTTCGTGCCCCCGCCGT20     (2) INFORMATION FOR SEQ ID NO:57:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:     TGCTGTTCGTGCCCCCGCCG20     (2) INFORMATION FOR SEQ ID NO:58:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:     ATGCTGTTCGTGCCCCCGCC20     (2) INFORMATION FOR SEQ ID NO:59:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:     GATGCTGTTCGTGCCCCCGC20     (2) INFORMATION FOR SEQ ID NO:60:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:     GGGCCAGGCTCACGCGCTGC20     (2) INFORMATION FOR SEQ ID NO:61:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:     GCCCGGGCCAGGCTCACGCG20     (2) INFORMATION FOR SEQ ID NO:62:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:     CCCTGGACCGCTGACGCCCG20     (2) INFORMATION FOR SEQ ID NO:63:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:     CGCCCGTGACCCCGTTCTCC20     (2) INFORMATION FOR SEQ ID NO:64:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:     GCGGGATGATGATGGCGGCG20     (2) INFORMATION FOR SEQ ID NO:65:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:     CGGGATGATGATGGCGGCGA20     (2) INFORMATION FOR SEQ ID NO:66:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:     GGCGGGATGATGATGGCGGC20     (2) INFORMATION FOR SEQ ID NO:67:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:     GGGCGGGATGATGATGGCGG20     (2) INFORMATION FOR SEQ ID NO:68:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:     GGGGCGGGATGATGATGGCG20     (2) INFORMATION FOR SEQ ID NO:69:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:     AGGGGCGGGATGATGATGGC20     (2) INFORMATION FOR SEQ ID NO:70:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:     ATGGCGGCGATGGGCGTGGC20     (2) INFORMATION FOR SEQ ID NO:71:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:     GATGGCGGCGATGGGCGTGG20     (2) INFORMATION FOR SEQ ID NO:72:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:     TGATGGCGGCGATGGGCGTG20     (2) INFORMATION FOR SEQ ID NO:73:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:     ATGATGGCGGCGATGGGCGT20     (2) INFORMATION FOR SEQ ID NO:74:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:     GATGATGGCGGCGATGGGCG20     (2) INFORMATION FOR SEQ ID NO:75:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:     TGATGATGGCGGCGATGGGC20     (2) INFORMATION FOR SEQ ID NO:76:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 19 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:76:     CGATGCCGACCTTTTCTCC19     (2) INFORMATION FOR SEQ ID NO:77:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:77:     GCCCCACGATGCCGACCTTT20     (2) INFORMATION FOR SEQ ID NO:78:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:78:     CGCCCCACGATGCCGACCTT20     (2) INFORMATION FOR SEQ ID NO:79:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:79:     CCGCCCCACGATGCCGACCT20     (2) INFORMATION FOR SEQ ID NO:80:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:80:     TCCGCCCCACGATGCCGACC20     (2) INFORMATION FOR SEQ ID NO:81:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:81:     TGGCGGTGGCTGCTGCTTTG20     (2) INFORMATION FOR SEQ ID NO:82:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:82:     GGATGGCGGTGGCTGCTGCT20     (2) INFORMATION FOR SEQ ID NO:83:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:83:     CGGATGGCGGTGGCTGCTGC20     (2) INFORMATION FOR SEQ ID NO:84:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 22 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:84:     CCGGTGGGCGATGGTGAGGACG22     (2) INFORMATION FOR SEQ ID NO:85:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:85:     TGTCTCCGCTTCTTCCTGCC20     (2) INFORMATION FOR SEQ ID NO:86:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 15 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:86:     GCTCCTCCATTGCGG15     (2) INFORMATION FOR SEQ ID NO:87:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:87:     CCTCGGTCCCCCCTCGTCCC20     (2) INFORMATION FOR SEQ ID NO:88:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 25 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:88:     CCCCTGGCGTTACCTCCTCGTTTCT25     (2) INFORMATION FOR SEQ ID NO:89:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 25 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:89:     TTCGCCTGATTTCCGCCTCCCGTCT25     (2) INFORMATION FOR SEQ ID NO:90:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 18 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:90:     CGGTCCGTTATGTTCCTG18     (2) INFORMATION FOR SEQ ID NO:91:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 22 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:91:     ACTCGCCTCCCACGTAGTGCTT22     (2) INFORMATION FOR SEQ ID NO:92:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 22 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:92:     CGTGCCCCTACCTCGCGCTCCT22     (2) INFORMATION FOR SEQ ID NO:93:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 24 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:93:     CCCTACCTCGCGCTCCTTGGAACG24     (2) INFORMATION FOR SEQ ID NO:94:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 22 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (ix) FEATURE:     (A) NAME/KEY: misc.sub.-- feature     (B) LOCATION: 4TH POSITION     (D) OTHER INFORMATION: "oligonucleotide"     Inosine substitution for thymidine gives perfect     match with both bindi     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:94:     CCCNACCTCGCGCTCCTTGGAA22     (2) INFORMATION FOR SEQ ID NO:95:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 24 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:95:     CGTGCCCCTACCTCGCGCTCCTTG24     (2) INFORMATION FOR SEQ ID NO:96:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 18 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:96:     CGTGCCCCTACCTCGCGC18     (2) INFORMATION FOR SEQ ID NO:97:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 18 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:97:     TCCCGACCTCGCGCTCCT18     (2) INFORMATION FOR SEQ ID NO:98:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 22 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (ix) FEATURE:     (A) NAME/KEY: misc.sub.-- feature     (B) LOCATION: 4TH POSITION     (D) OTHER INFORMATION: Substitution of guanine with     inosine gives a perfect match with both binding sites.     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:98:     CCCNACCTCGCGCTCCTTGGAA22     (2) INFORMATION FOR SEQ ID NO:99:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 24 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:99:     CCATCCCGACCTCGCGCTCCTTGG24     (2) INFORMATION FOR SEQ ID NO:100:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 26 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:100:     CCATCCCGACCTCGCGCTCCTTGGAA26     (2) INFORMATION FOR SEQ ID NO:101:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (ix) FEATURE:     (A) NAME/KEY: misc.sub.-- feature     (B) LOCATION: 4TH POSITION     (D) OTHER INFORMATION: This variant sequence contains an     inosine base substituted at the fourth position where     the single base variation between SEQ ID:94 and SEQ ID     NO:98 exists.     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:101:     CCCNACCTCGCGCTCCTTGG20     (2) INFORMATION FOR SEQ ID NO:102:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 38 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: NO     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:102:     TGAAGGGGCAAGCAATGGAGGAGCAAAGAAGAAGAACT38     (2) INFORMATION FOR SEQ ID NO:103:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 25 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: NO     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:103:     GGAAGCCTGAGCTCATTCGAGTAGC25     (2) INFORMATION FOR SEQ ID NO:104:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 23 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: NO     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:104:     AGGGGCACGCAAAGCTGGGAGCT23     (2) INFORMATION FOR SEQ ID NO:105:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 24 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: NO     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:105:     GGACCATGTTGCCCGGAGCGCGCA24     (2) INFORMATION FOR SEQ ID NO:106:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 39 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: NO     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:106:     GCCACGCCCATCGCCGCCATCATCATCCCGCCCCTTGGC39     (2) INFORMATION FOR SEQ ID NO:107:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 70 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: NO     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:107:     CGGACAGAGATTGGCGAGAAGGGCGTGAACCTGTCTGGGGGCCAGAAGCA50     GCGCGTGAGCCTGGCCCGGG70     (2) INFORMATION FOR SEQ ID NO:108:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 45 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: NO     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:108:     TCCACGACCTGATGATGTTTTCCGGGCCGCAGATCTTAAAGTTGC45     (2) INFORMATION FOR SEQ ID NO:109:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 90 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: NO     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:109:     GCCAGCACAGAGCAGGAGCAGGATGCAGAGGAAAGGGGTCACGGGCGTCA50     GCGGTCCAGGGAAGGAAGCAAAGCAAATGGAGAATGGGAT90     (2) INFORMATION FOR SEQ ID NO:110:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (ix) SEQUENCE DESCRIPTION: SEQ ID NO:110:     TAGCCACATGGCCCCAGGAA20     (2) INFORMATION FOR SEQ ID NO: 111:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 111:     ACTGACTTGCCCCACGGCCA20     (2) INFORMATION FOR SEQ ID NO: 112:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (ix) SEQUENCE DESCRIPTION: SEQ ID NO:112:     CCAAAGGGCAAAGGGCAAGG20     (2) INFORMATION FOR SEQ ID NO: 113:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 113:     GTACCTTACCTTTTATCTGG20     (2) INFORMATION FOR SEQ ID NO: 114:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: Not Relevant     (ii) MOLECULE TYPE: DNA (genomic)     (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: YES     (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 114:     TGCCCCTACCTCGCGCTCCT20     __________________________________________________________________________ 

What is claimed is:
 1. A modified oligonucleotide between 15 and 30 nucleotides in length, inclusive, having a sequence that specifically hybridizes in a human cell with a complementary sequence of a human MDR1 gene and allelic variants thereof to inhibit expression of a multidrug resistance phenotype exhibited by said cell, said complementary sequence being selected from the group consisting of SEQ ID Nos: 103, 104 and 105 and said modification being a backbone modification selected from the group consisting of dithioate, methylphosphonate, morpholino, polyamide, or any combination of said modification.
 2. The oligonucleotide of claim 1, which is between 17 and 26 nucleotides in length.
 3. The oligonucleotide of claim 1, wherein said backbone modification further includes a phosphorothioate-modification.
 4. The modified oligonucleotide of claim 1, which specifically hybridizes to said MDR1 gene and has a sequence selected from the group consisting of SEQ ID Nos: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 35, 36, 37, 38, 39, 40, 92, 93, 94, 95, 96, 97, 98, 99, 100 and
 101. 5. The modified oligonucleotide of claim 1, said oligonucleotide further comprising at least one 2'O-methyl ribonucleoside modification.
 6. A modified oligonucleotide between 15 and 30 nucleotides in length, inclusive, consisting of a sequence that specifically hybridizes in a human cell with a complementary sequence of a human MRP gene and allelic variants thereof to inhibit expression of a multidrug resistance phenotype exhibited by said cell, said complementary sequence being selected from the group consisting of Sequence ID Nos: 106, 107, 108 and 109 and said modification being a backbone modification selected from the group consisting of dithioate, methylphosphonate, morpholino, polyamide, or any combination of said modification.
 7. The modified oligonucleotide of claim 6, which is between 17 and 26 nucleotides in length, inclusive.
 8. The modified oligonucleotide of claim 6, wherein said backbone modification further includes a phosphorothioate-modification.
 9. The modified oligonucleotide of claim 6, consisting of a sequence which specifically hybridizes with said MRP gene and allelic variants thereof selected from the group consisting of Sequence ID Nos: 42, 43, 44, 45, 46, 47, 48, 49, 60, 62, 64 and
 76. 10. The modified oligonucleotide of claim 6, said oligonucleotide further comprising at least one 2'O-methyl ribonucleoside modification.
 11. A modified oligonucleotide between 17 and 30 nucleotides in length, inclusive, having a sequence that specifically hybridizes in a human cell with a complementary sequence of a human MDR1 gene and allelic variants thereof to inhibit expression of a multidrug resistance phenotype exhibited by said cell, said complementary sequence having the sequence of Sequence ID No: 102 and said modification being a backbone modification selected from the group consisting of dithioate, methylphosphonate, morpholino, polyamide, or any combination of said modification.
 12. The modified oligonucleotide of claim 11, which is between 17 and 26 nucleotides in length, inclusive.
 13. The modified oligonucleotide of claim 11, wherein said backbone modification further includes a phosphorothioate-modification.
 14. The modified oligonucleotide of claim 11, which specifically hybridizes to said MDR1 gene, consisting of a sequence selected from the group consisting of Sequence ID Nos: 25, 26, 27, 28, 29, 30, 31, 32, 33 and
 34. 15. The modified oligonucleotide of claim 11, said oligonucleotide further comprising at least one 2'O-methyl ribonucleoside modification. 